We recently identified the genotoxic bifunctional electrophiles, 4,5-epoxy-2-decenal, 4-oxo-2-nonenal, and 4- hydroxy-2-nonenal together with the potential genotoxin 4- hydroperoxy-2-nonenal as major products of vitamin C-mediated decomposition of lipid hydroperoxides. Surprisingly, vitamin C was more than twice as efficient as transition metal ions at inducing this decomposition. It has long been thought that lipid hydroperoxides play a role in degenerative diseases of aging such as cancer and cardiovascular disease. We have now made the exciting observation that 4-oxo-2-nonenal efficiently forms adducts with the amino acid arginine. Previous studies have demonstrated 4-hydroxy-2-nonenal can induce covalent modifications with model amino acids. However, the efficiency of this process is much lower than we observed with 4-oxo-2-nonenal The ability to quantify covalent modifications to plasma amino acids, plasma proteins, and arginine-rich histones will provide a dosimeter of exposure to lipid hydroperoxide-derived genotoxins that would complement data obtained with DNA-adducts. Furthermore, quantitation of covalent modifications to arginine and arginine-rich histones could provide additional insight into cancer risk. Plasma arginine is the precursor for the generation a nitric oxide a known mediator of tumorigenesis. Lipid hydroperoxide-mediated covalent modification of free plasma arginine would prevent deiminase-mediated conversion to citrulline and nitric oxide. This would limit endogenous substrate availability and could also act as an endogenous inhibitor. Endogenous inhibitors have been proposed but none have not been detected in sufficiently large amounts to account for the L-arginine paradox. This paradox arises from the observation that infusions of L-arginine can cause additional nitric oxides release even though there is apparently sufficient free intracellular arginine to saturate endogenous nitric oxide synthases. Covalent modifications to histone arginines would also affect transcription and cellular proliferation. We propose to focus initially on quantifying the covalent modifications that are induced by the interaction of lipid hydroperoxide-derived bifunctional electrophiles with free arginine. Studies will then be conducted with hemoglobin, albumin, and histones in vitro. Structural analysis will be performed using conventional protease digests coupled with liquid chromatography and tandem mass spectrometry. If necessary, specific covalent modifications will be characterized by chemical synthesis. The relative importance of these lesions will then be assessed when the bifunctional electrophiles are generated by vitamin C-mediated homolytic decomposition of lipid hydroperoxides. Such studies have been performed in the past using transition metal ion-mediated decomposition of lipid hydroperoxides. Unfortunately, transition metal ions also catalyze Huber-Weiss reactions, which results in the formation of reactive oxygen species. The use of transition metal ion-free buffers and vitamin C overcomes this problem. Therefore, it will be possible for the first time to study lipid hydroperoxide-mediated protein damage without the complication of simultaneous oxidative damage. The changes in histone function resulting from vitamin C-induced lipid hydroperoxide decomposition will be assessed in relevant in vitro models. Finally, free arginine, albumin and hemoglobin will be isolated from plasma of patients with leukemia. Lipid hydroperoxide- derived covalent modifications in arginine and proteins from these populations will then be quantified by stable isotope dilution liquid chromatography/tandem mass spectrometry and compared with arginine and proteins from normal subjects. These studies will serve as a precursor to future dosimetry studies in large populations and will provide insight into the role of lipid peroxidation as a mediator of carcinogenesis.